Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface (original) (raw)
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BioMed research international, 2013
The EU Regulation on Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) demands the implementation of alternative methods for analyzing the hazardous effects of chemicals including particulate formulations. In the field of inhalation toxicology, a variety of in vitro models have been developed for such studies. To simulate the in vivo situation, an adequate exposure device is necessary for the direct exposure of cultivated lung cells at the air-liquid interface (ALI). The CULTEX RFS fulfills these requirements and has been optimized for the exposure of cells to atomized suspensions, gases, and volatile compounds as well as micro- and nanosized particles. This study provides information on the construction and functional aspects of the exposure device. By using the Computational Fluid Dynamics (CFD) analysis, the technical design was optimized to realize a stable, reproducible, and homogeneous deposition of particles. The efficiency of the exposure procedure...
Toxicology in Vitro, 2019
The CULTEX ® Radial Flow System (RFS) is a modular in vitro system for the homogenous exposure of cells to airborne particles at the air-liquid interface (ALI). A former prevalidation study successfully demonstrated the general applicability of the CULTEX ® RFS and its transferability, stability and reproducibility. Based on these results, the methodology was optimized, validated and prediction models for acute inhalation hazards were established. Cell viability of A549 cells after ALI exposure to 20 pre-selected test substances was assessed in three independent laboratories. Cytotoxicity of test substances was compared to the respective incubator controls and used as an indicator of toxicity. Substances were considered to exert an acute inhalation hazard when viability decreased below 50% (prediction model (PM) 50%) or 75% (PM 75%) at any of three exposure doses (25, 50 or 100 µg/cm²). Results were then compared to existing in vivo data and revealed an overall concordance of 85%, with a specificity of 83% and a sensitivity of 88%. Depending on the applied PM, the withinlaboratory and between-laboratory reproducibility ranged from 90-100%. In summary, the CULTEX ® RFS was proven as a transferable, reproducible and well predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles.
Journal of Aerosol Science, 2016
In vitro toxicity testing of airborne particles usually takes place in multi-well plates, where the cells are exposed to a suspension of particles in cell culture medium. Due to the artefacts caused by particle collection and preparation of suspensions, the air-liquid interface (ALI) exposure is challenging this conventional exposure technique to become the method of choice. The ALI technique allows for direct sampling of an aerosol and exposure of cell cultures to airborne particles. At the same time, it reflects the physiological conditions in the lung to a greater extent. So far, the available ALI systems have mostly been laboratory setups of the single components. Here, we present a mobile and complete system providing all process technology required for cell exposure experiments at dynamic aerosol sources. The system is controlled by a human machine interface (HMI) with standard routines for experiments and internal testing to assure reproducibility. It also provides documentation of the exposure experiment regarding process parameters and measured doses. The performance of this system is evaluated using fluoresceinsodium dosimetry, which is also used to determine the factor of dose enhancement by optional electrostatic deposition. The application of the system is shown for two different technical aerosol sources: wood smoke particles emitted by a household log wood stove Contents lists available at ScienceDirect
Direct and indirect air particle cytotoxicity in human alveolar epithelial cells
Toxicology in Vitro, 2014
Air particulate matter has been associated with adverse impact on the respiratory system leading to cytotoxic and proinflammatory effects. The biological mechanisms behind these associations may be initiated by inhaled small size particles, particle components (soluble fraction) and/or mediators released by particle-exposed cells (conditioned media). The effect of Urban Air Particles from Buenos Aires (UAP-BA) and Residual Oil Fly Ash (ROFA) a surrogate of ambient air pollution, their soluble fractions (SF) and conditioned media (CM) on A549 lung epithelial cells was examined. After 24h exposure to TP (10 and 100 µg/ml), SF or CM, several biological parameters were assayed on cultured A549 cells. We tested cell viability by MTT, superoxide anion (O 2-) generation by NBT and proinflammatory cytokine (TNF α , IL-6 and IL-8) production by ELISA. UAP-BA particles or its SF (direct effect) did not modify cell viability and generation of O 2 for any of the doses tested. On the contrary, UAP-BA CM (indirect effect) reduced cell viability and increased both generation of O 2 and IL-8 production. Exposure to ROFA particles, SF or ROFA CM reduced proliferation and O 2 but, stimulated IL-8. It is worth to note that UAP-BA and ROFA depicted distinct effects on particle-exposed A549 cells implicating morphochemical dependence. These in vitro findings support the hypothesis that particle-induced lung inflammation and disease may involve lung-derived mediators.
Alternatives to Laboratory Animals, 2002
The application of in vitro methods to the analysis of the effects of airborne materials is still limited, because there are no generally accepted concepts and technologies for efficiently exposing adherent growing cells to test atmospheres, especially those comprising complex mixtures of gaseous and particulate phases. The introduction of in vitro research into the field of inhalation toxicology offers a unique possibility for using human cells and tissues for pre-screening studies, thus reducing the necessity for animal experiments, and cutting the numbers of animals used in toxicological testing. We therefore developed a novel experimental concept that uses an exposure device based on the cell cultivation system CULTEX (Patent No. DE 198011763; PCT/EP99/00295). This allowed us to investigate environmental atmospheres, which were chemically and physically unmodified, in an in vitro system, by exposing the target cells directly at the air/liquid interface. The exposure device itsel...
Background: Exposure to fine and ultra-fine ambient particles is still a problem of concern in many industrialised parts of the world and the intensified use of nanotechnology may further increase exposure to small particles. Complex in vitro coculture systems may be valuable tools to study particle-induced processes and to extrapolate effects of particles on the lung. A system consisting of four different human cell lines which mimics the cell response of the alveolar surface in vitro was developed to study native aerosol exposure (Vitrocell™ chamber). The system is composed of an alveolar type-II cell line (A549), differentiated macrophage-like cells (THP-1), mast cells (HMC-1) and endothelial cells (EA.hy 926), seeded in a 3D-orientation on a microporous membrane. Results: The spatial distribution of the cells in the tetraculture was analysed by confocal laser scanning microscopy (CLSM), showing a confluent layer of endothelial and epithelial cells on both sides of the transwell. Macrophage-like cells and mast cells can be found on top of the epithelial cells. The cells formed colonies under submerged conditions, which disappeared at the ALI. To evaluate the response to oxidative stress, the dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used together with 2,2'-azobis-2-methylpropanimidamide-dihydrochloride (AAPH) as inducer of oxidative stress. The tetraculture showed less induction of reactive oxygen species (ROS) production after being treated with a positive control compared to the monocultures of EA.hy 926, THP-1 and HMC-1. Submerged cultures showed elevated ROS and IL-8 levels compared to ALI cultures. The Vitrocell™ aerosol exposure system was not significantly influencing the viability. Using this system, cells were exposed to an aerosol of 50 nm SiO 2-Rhodamine NPs in PBS. The distribution of the NPs in the tetraculture after exposure was evaluated by CLSM. Fluorescence from internalized particles was detected in CD11b-positive THP-1 cells only. Conclusion: The system can be used in conjunction with a native aerosol exposure system and may finally lead to a more realistic judgement regarding the hazard of new compounds and/or new nano-scaled materials in the future. The results for the ROS production and IL-8 secretion suggest that submerged exposure may lead to an overestimation of observed effects.
Journal of Aerosol Science, 2021
Inhalation exposure to environmental and occupational aerosol contaminants is associated with many respiratory health problems. To realistically mimic long-term inhalation exposure for toxicity testing, lung epithelial cells need to maintained and exposed under air-liquid interface (ALI) conditions for a prolonged period of time. In addition, to study cellular responses to aerosol particles, lung epithelial cells have to be co-cultured with macrophages. To that aim, we evaluated human bronchial epithelial Calu-3, 16HBE14o-(16HBE), H292, and BEAS-2B cell lines with respect to epithelial morphology, barrier function and cell viability under prolonged ALI culture conditions. Only the Calu-3 cells can retain the monolayer structure and maintain a strong tight junction under long-term ALI culture at least up to 2 weeks. As such, Calu-3 cells were applied as the structural barrier to create co-culture models with human monocyte-derived macrophages (MDMs) and THP-1 derived macrophages (TDMs). Adhesion of macrophages onto the epithelial monolayer was allowed for 4 h with a density of 5 × 10 4 macrophages/cm 2. In comparison to the Calu-3 mono-culture model, Calu-3 + TDM and Calu-3 + MDM co-culture models showed an increased sensitivity in inflammatory responses to lipopolysaccharide (LPS) aerosol at Day 1 of co-culture, with the Calu-3 + MDM model giving a stronger response than Calu-3 + TDM. Therefore, the epithelial monolayer integrity and increased sensitivity make the Calu-3 + MDM co-culture model a preferred option for ALI exposure to inhaled aerosols for toxicity testing.
In situ-Like Aerosol Inhalation Exposure for Cytotoxicity Assessment Using Airway-on-Chips Platforms
Frontiers in Bioengineering and Biotechnology, 2020
Lung exposure to inhaled particulate matter (PM) is known to injure the airway epithelium via inflammation, a phenomenon linked to increased levels of global morbidity and mortality. To evaluate physiological outcomes following PM exposure and concurrently circumvent the use of animal experiments, in vitro approaches have typically relied on traditional assays with plates or well inserts. Yet, these manifest drawbacks including the inability to capture physiological inhalation conditions and aerosol deposition characteristics relative to in vivo human conditions. Here, we present a novel airway-onchip exposure platform that emulates the epithelium of human bronchial airways with critical cellular barrier functions at an air-liquid interface (ALI). As a proof-of-concept for in vitro lung cytotoxicity testing, we recapitulate a well-characterized cell apoptosis pathway, induced through exposure to 2 µm airborne particles coated with αVR1 antibody that leads to significant loss in cell viability across the recapitulated airway epithelium. Notably, our in vitro inhalation assays enable simultaneous aerosol exposure across multiple airway chips integrated within a larger bronchial airway tree model, under physiological respiratory airflow conditions. Our findings underscore in situ-like aerosol deposition outcomes where patterns depend on respiratory flows across the airway tree geometry and gravitational orientation, as corroborated by concurrent numerical simulations. Our airway-on-chips not only highlight the prospect of realistic in vitro exposure assays in recapitulating characteristic local in vivo deposition outcomes, such platforms open opportunities toward advanced in vitro exposure assays for preclinical cytotoxicity and drug screening applications.